Influenza viruses continue to pose a significant threat to global public health. Consequently, there is a vitally important need to generate new influenza virus vaccines and innovative vaccination strategies. The development of a long-lived broadly-protective influenza virus vaccine would have a tremendous impact on worldwide efforts to control influenza viruses. The purpose of this application is to accelerate the development of a universal influenza virus vaccine through the preclinical development of two lead candidate components of such a vaccine. Since our initial discovery that sequential immunization with influenza vaccines based on chimeric HAs induce broad protection against diverse influenza virus strains in animal models, we have moved into Phase 1 clinical trials that investigate the safety and immunogenicity of inactivated adjuvanted and cold-adapted live attenuated influenza A virus vaccines containing chimeric group 1 influenza virus HAs. Such vaccines induce protective immunity in mice, ferrets and pigs against all group 1 influenza A virus, including H1, H2 and H5 viruses. Protection correlates with the induction of high levels of cross-reactive Fc-receptor engaging antibodies against the conserved group 1 HA stalk. If any of these vaccination strategies are successful in humans, addition of group 2 influenza A and influenza B chimeric HAs would result in protection against any type, subtype and strain of influenza virus. This ?universal flu vaccine? would eliminate the need for annual influenza virus vaccination and prevent future influenza pandemics. In the current application we propose to generate GMP clinical lots and IND-enabling information to subsequently perform human clinical trials with a highly immunogenic vaccine platform expressing chimeric HAs based on live attenuated delNS1 influenza viruses. In contrast to the cold-adapted influenza vaccines, delNS1 influenza vaccines are highly immunostimulatory due to the removal of the immune antagonist gene NS1. For the purpose of this grant, our group at Icahn School of Medicine at Mount Sinai, which pioneered the use of chimeric HAs for universal influenza virus vaccination, has partnered with Vivaldi, a biotechnology company that has delNS1-based influenza virus vaccines under clinical development. At the end of our proposal, we will have generated two GMP lots of delNS1-based group 1 chimeric HA influenza virus vaccines ready for clinical investigation. These lots will be used after completion of our grant for sequential immunizations in humans to obtain their safety and immunogenicity profiles. Moreover, we anticipate that human influenza virus challenge studies after vaccination with inactivated, cold-adapted and delNS1-based chimeric HA vaccines will differentiate the vaccine platform among these three resulting in best levels of protection. The best platform will be used as the basis for further clinical development and incorporation of group 1, group 2 and influenza B chimeric HAs to protect against all possible influenza viruses.